CN115669921A - Liver-protecting and kidney-protecting cistanche enzyme and preparation method thereof - Google Patents
Liver-protecting and kidney-protecting cistanche enzyme and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a cistanche enzyme for protecting liver and kidney and a preparation method thereof, wherein cistanche is taken as a raw material, cistanche is extracted, and is mixed according to a proportion to carry out enzymolysis, and lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei are inoculated to prepare mixed bacteria for fermentation after the enzymolysis is finished, so that the cistanche enzyme for protecting liver and kidney is finally obtained.
Description
Technical Field
The invention belongs to the technical field of food processing enzymes, and particularly relates to a cistanche enzyme for protecting liver and kidney and a preparation method thereof.
Background
The plant enzyme is a functional product which is obtained by taking one or more fresh fruits, vegetables, cereals, mushrooms or other medicinal and edible herbs as a fermentation raw material through microbial fermentation and is rich in various nutritional and bioactive substances, such as enzymes, polyphenol compounds, minerals, organic acids and the like. The ferment is a well-known pure natural health product, and microorganisms in the ferment can not only produce secondary metabolites such as various functional enzymes, amino acids, short-chain fatty acids, alcohols, esters and the like, but also can convert certain components of fermentation raw materials into high-activity substances in the fermentation process. The enzyme produced by fermenting the beneficial microorganisms has increased superoxide dismutase (SOD) activity and better antioxidant capacity. The fruit and vegetable processing in China is mainly fruit and vegetable juice beverage processing, and new types of fruit and vegetable enzyme beverages are gradually developed at present, but the domestic plant enzyme products are relatively single in type and different in quality, and the research on the complex fermentation process and processing technology is not deep enough, for example, the research on cistanche enzyme is few.
Cistanche, also known as golden bamboo shoot, herba violae, cistanches and arum chinensis, is a dry fleshy stem of cistanche deserticola which is a perennial parasitic herbaceous plant of Orobanchaceae, mostly grows from the roots of Haloxylon H.amodentron and Haloxylon H.persicum of Chenopodiaceae, has strong drought resistance, cold resistance and salt and alkali resistance, is a famous Chinese medicinal material in China, and has the name of desert ginseng. The modern pharmacology proves that the cistanche deserticola contains various medicinal components, has various biological activities of resisting liver injury, resisting tumors, resisting viruses, reducing blood sugar, resisting blood coagulation, resisting oxidation, regulating immunity and the like, and has extremely high medicinal value. At present, the research on cistanche is mostly focused on the extraction pharmacological action of the effective chemical components and the development and application of related products thereof. Is relatively deficient in the development and application in the fields of new drugs, health products, foods, drinks and the like. The products which are developed and circulated in the market at present comprise medicaments and health-care products such as cistanche deserticola wine, cistanche deserticola health-preserving liquid, kidney-nourishing liquid, cistanche deserticola oral liquid, cistanche deserticola tea, cistanche deserticola capsules, cistanche deserticola tablets, cistanche deserticola granules, cistanche deserticola decocted pills and the like. Cistanche is also commonly used as food and health care products to appear in the diet of people, and the common cistanche is cistanche congee, cistanche edestan paste, cistanche wine, cistanche medlar wine, cistanche dodder wine and the like. Therefore, the intensive research on the cistanche deserticola is carried out, the medicinal health care value of the cistanche deserticola is developed, the value of the cistanche deserticola is increased, and the research is a new opportunity for the production and research institutions of the cistanche deserticola.
In addition, the cistanche ferment reported in the Chinese patent CN110250507A is prepared by mixing the cistanche with fruits and Chinese herbal medicines and then adopting an extraction and fermentation combined process to improve the utilization rate of the cistanche, and the obtained ferment SOD activity is 1100-1300U/g. Chinese patent CN109601799A reports a preparation method of cistanche ferment beverage, which comprises inoculating rhizopus and red yeast rice strains, sealing and fermenting for 3-6 months to obtain the beverage.
Although cistanche ferment is reported, in order to improve the utilization rate of cistanche, the main raw materials of cistanche and other fruits or traditional Chinese medicinal materials are adopted, but the adopted strains are complex, the key of the liquid submerged fermentation technology lies in a culture medium, different edible fungi need to be cultured by different culture media, fermentation and environmental conditions need to be considered, and the fermentation control links are multiple and complex and are difficult to control. Meanwhile, the fermentation stage needs to be carried out in stages, the first stage is an aerobic stage, the ventilation amount of sterile air needs to be controlled, the second stage is an anaerobic stage, ventilation needs to be closed, and the fermentation temperature needs to be controlled, so that the preparation process is complex, the possibility of contaminating mixed bacteria is increased, the fermentation period is long, and the preparation cost is increased. And the existing patents and documents do not report the cistanche ferment for protecting the liver and the kidney by fermenting cistanche serving as a raw material.
Disclosure of Invention
Aiming at the problems that the existing cistanche ferment preparation process is complex in preparation process and difficult in screening of prepared strains caused by extraction and fermentation of fruits or traditional Chinese medicines as main raw materials, different strains need to be cultured by adopting different culture media, fermentation, environmental conditions and the like need to be considered, the fermentation control links are multiple and complex, the actual situation is difficult to control, and the technical current situation of preparing the cistanche ferment for protecting the liver and the kidney by directly fermenting the cistanche is not reported in patents and literatures. The invention aims to provide a cistanche enzyme for protecting liver and kidney and a preparation method thereof. The SOD activity is improved to 625.03U/mL from the original 401.26U/mL, the clearance rate of hydroxyl radical reaches 65.02%, the clearance rate of superoxide anion reaches 85.98%, the clearance rate of DPPH radical reaches 93.68%, the flavor of cistanche is improved by fermentation, the cistanche ferment has special sour taste due to a large amount of organic acid generated in the fermentation process, the whole cistanche ferment is sour, sweet and delicious, the sensory score is improved to 94.00 +/-0.77 from 84.10 +/-1.14, and the cistanche ferment has extremely high edible and health-care values. Therefore, the cistanche ferment has great market potential, and the application range of cistanche is expanded to a certain extent, so that the cistanche which is a valuable resource has more considerable development prospect.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides cistanche ferment with liver and kidney protecting effects, and a preparation method of the ferment specifically comprises the following steps:
(1) Respectively pouring the obtained freeze-dried powder of lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei on a sterile operation table into a conical flask filled with a sterile MRS liquid culture medium, sealing the conical flask, placing the conical flask into a constant-temperature incubator at 37 ℃ for activation for 24 to 48 hours to obtain bacterial suspension, counting the bacterial suspension by using a blood counting plate under a microscope, wherein the bacterial density of the bacterial suspension is about 10 8 CFU/mL, after which culture 2 is continued-3 for fermentation, the corresponding bacterial suspension obtained is stored in a refrigerated cabinet at 4 ℃ for subsequent use.
(2) Weighing and cleaning the selected cistanche slices, and mixing the materials in percentage by volume: water =1: adding water in a proportion of 7-10, decocting for 30-60 min, cooling and pulping to obtain the cistanche salsa serous fluid.
(3) And (3) taking the cistanche deserticola serous fluid obtained in the step (2), adding 0.4-0.8% of pectinase, 0.2-0.4% of cellulase and 0.2-0.4% of hemicellulase which are based on the weight of the cistanche deserticola slices, fully stirring, and carrying out enzymolysis for 2-3.5 h at the temperature of 50-60 ℃. Determining the content of soluble solid matters in the cistanche enzymolysis liquid after the enzymolysis reaction is finished; regulating sugar degree of the cistanche enzymolysis liquid to ensure that the sugar Brix is 9-13 degrees Bx.
(4) And (4) inactivating enzyme and sterilizing the cistanche enzymolysis liquid obtained in the step (3) at the temperature of 80-100 ℃ for 10-15 min, and then cooling to room temperature for later use.
(5) Inoculating the bacterial suspensions obtained in the step (1) according to a certain volume ratio of 2 x 10 6 ～5*10 6 And (3) CFU/mL, inoculating the obtained product into the cistanche enzymolysis liquid sterilized in the step (4) for fermentation at the fermentation temperature of 30-37 ℃ for 12-36 h, and measuring the SOD activity after the fermentation is finished, wherein the SOD activity is not lower than 486U/mL.
(6) Filtering the cistanche fermentation liquor obtained in the step (5) at a low temperature of 18-25 ℃, sterilizing at a temperature of 100-121 ℃ for 20-30 min, filling, sealing, packaging, and storing at a low temperature to obtain the finished cistanche ferment.
The invention provides a preparation method of cistanche enzyme for protecting liver and kidney, wherein bacterial suspension is obtained by mixing 30-34%, 14-18%, 15-19%, 10-14%, 12-16% and 7-11% of bacterial suspension of lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei according to the volume ratio, and the viable count of the bacterial suspension is more than or equal to 10 8 CFU/mL。
In the preparation of the liver and kidney protecting cistanche enzyme provided by the invention, the culture time of the strain is 36h, and the culture temperature is 37 ℃.
The invention provides a preparation method of cistanche deserticola ferment for protecting liver and kidney, wherein the volume ratio of cistanche deserticola to decoction water is 1:8, decocting for 40min.
The invention provides a preparation method of cistanche enzyme for protecting liver and kidney, wherein the enzymes are pectinase, cellulase and hemicellulase, the addition amounts of the enzymes are 0.6%, 0.3% and 0.3% of the weight of cistanche slices respectively, the enzymolysis time is 3.0h, and the enzymolysis temperature is 55 ℃.
In the preparation of the cistanche enzyme for protecting the liver and the kidney, the fermentation temperature is 32 ℃, and the fermentation time is 24 hours.
The invention provides a preparation method of cistanche enzyme for protecting liver and kidney, wherein the sterilization and enzyme deactivation temperature is 90 ℃, and the sterilization and enzyme deactivation time is 12min.
The invention also provides application of the cistanche ferment in liver and kidney protection.
Through the technical scheme, the invention achieves the following technical effects:
(1) The cistanche ferment prepared by the invention has higher content of functional components, and simultaneously adopts a microbial production method to produce superoxide dismutase (SOD) with the SOD of 625.03U/mL, so that the flavor and taste of cistanche are improved, organic acid and aromatic substances are generated, and the sensory score is 94.00 +/-0.77. Meanwhile, the clearance rate of hydroxyl radicals reaches 65.02 percent, the clearance rate of superoxide anions reaches 85.98 percent, and the clearance rate of DPPH radicals reaches 93.68 percent, which are obviously improved compared with the clearance rate of non-inoculated fermentation. Because oxidative stress is a negative effect generated by free radicals in vivo and is also considered as an important factor causing aging and diseases, the cistanche ferment prepared by the invention has the effects of nourishing heart, dredging collaterals, protecting liver and kidney, dredging intestinal tracts, resisting aging, resisting oxidation, regulating immunity and the like to a certain extent.
(2) The cistanche ferment provided by the invention does not need to be subjected to compound fermentation with fruits or traditional Chinese medicines to improve the flavor, the preparation process is simple and easy to operate, the flavor and the taste of cistanche are improved, the cistanche ferment is rich in superoxide dismutase and has the effects of protecting liver and kidney, dredging intestinal tract, resisting aging and oxidation and the like, the method not only widens the path of deep processing of cistanche, but also increases the new variety of the ferment, and has the typical characteristics of a new product.
(3) Although lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei are purchased from public sources, if the common culture medium of the six strains is applied to the implementation of the method, the fermentation and environmental conditions of cistanche ferment need to be considered, the fermentation control links are many and complicated, and the actual situation of difficult control is not easily realized, the prepared cistanche ferment cannot well reflect the SOD content of the prepared cistanche ferment, and the clearance rate of the prepared cistanche ferment on hydroxyl radicals, the clearance rate of superoxide anions and the DPPH radical are not as good as the effect obtained by adopting the unified preparation method provided by the invention.
Drawings
FIG. 1 is a graph showing the effect of the water decoction time and the enzymolysis time of cistanche ferment on the activity of cistanche ferment SOD.
FIG. 2 is a graph showing the effect of the water decoction time and fermentation time of cistanche ferment on the SOD activity of cistanche ferment in accordance with the present invention.
FIG. 3 is a graph showing the effect of the cistanche ferment enzymolysis time and fermentation time on the cistanche ferment SOD activity.
Fig. 4 is a graph showing the influence of the water decoction time and the enzymolysis time of cistanche ferment on the sensory score of cistanche ferment in the present invention.
Fig. 5 is a graph showing the influence of the water decoction time and fermentation time of cistanche ferment on the sensory score of cistanche ferment in accordance with the present invention.
FIG. 6 is a graph showing the effect of the fermentation time and enzymolysis time of cistanche ferments on sensory evaluation of cistanche ferments.
FIG. 7 is a graph showing the effect of different concentrations of cistanche ferments on cell viability.
FIG. 8 is a graph showing the effect of various concentrations of cistanche ferments on the survival rate of alcohol-damaged cells.
Detailed Description
Preferred embodiments of the present invention are described in detail below. Although preferred embodiments of the present invention are described below, it should be understood by those skilled in the art that the present invention is not limited to the embodiments illustrated herein and that modifications or substitutions in the details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, which falls within the scope of the present invention.
The lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei adopted by the invention can be purchased and obtained from public channels.
Example 1: preparation of cistanche enzyme for protecting liver and kidney
The embodiment provides a cistanche enzyme for protecting liver and kidney, which is obtained by the following preparation method:
(1) Respectively pouring the obtained freeze-dried powder of lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei on a sterile operation table into a conical flask filled with a sterile MRS liquid culture medium, sealing the conical flask, placing the conical flask into a constant-temperature incubator at 37 ℃ for activation for 24 to 48 hours to obtain bacterial suspension, counting the bacterial suspension by using a blood counting plate under a microscope, wherein the bacterial density of the bacterial suspension is about 10 8 CFU/mL, then continuing to culture 2-3 for fermentation, and storing the prepared corresponding bacterial suspension in a refrigerated cabinet at 4 ℃ for later use.
(2) Weighing and cleaning the selected cistanche slices, and mixing the materials in percentage by volume: water =1: adding water in a proportion of 7-10, decocting for 30-60 min, cooling and pulping to obtain the cistanche salsa serous fluid.
(3) And (3) taking the cistanche salsa serous fluid obtained in the step (2), adding 0.4-0.8% of pectinase, 0.2-0.4% of cellulase and 0.2-0.4% of hemicellulase in weight of the cistanche salsa slices, fully stirring, and carrying out enzymolysis for 2-3.5 h at 50-60 ℃. Determining the content of soluble solid matters in the cistanche enzymolysis liquid after the enzymolysis reaction is finished; regulating sugar degree of herba cistanches enzymolysis solution to make sugar Brix degree 9-13 ° Brix degree (Bx).
(4) And (4) inactivating enzyme and sterilizing the cistanche enzymolysis liquid obtained in the step (3) at the temperature of 80-100 ℃ for 10-15 min, and then cooling to room temperature for later use.
(5) Inoculating the bacterial suspensions obtained in the step (1) with the inoculum size of 2 x 10 according to a certain volume ratio 6 ～5*10 6 CFU/mL, inoculating to the sterilized herba cistanches enzymolysis liquid in the step (4)Fermenting at 30-37 deg.c for 12-36 hr, and measuring SOD activity not lower than 486U/mL.
(6) Filtering the cistanche fermentation liquor obtained in the step (5) at the low temperature of 18-25 ℃, sterilizing at the temperature of 100-121 ℃ for 20-30 min, filling, sealing, packaging and storing at the low temperature to obtain the finished cistanche ferment.
The bacterial suspension is obtained by mixing 30-34 percent, 14-18 percent, 15-19 percent, 10-14 percent, 12-16 percent and 7-11 percent of bacterial suspension of lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei according to the volume ratio, and the viable count of the bacterial suspension is more than or equal to 10 8 CFU/mL。
Example 2: preparation of cistanche enzyme for protecting liver and kidney
In this embodiment, on the basis of embodiment 1, a method for preparing cistanche enzyme for protecting liver and kidney is provided, wherein a volume ratio of cistanche to boiling water is 1:8, decocting for 40min; pectinase, cellulase and hemicellulase, wherein the addition amount of the enzymes is 0.6%, 0.3% and 0.3% of the weight of the cistanche slice respectively, the enzymolysis time is 3.0h, the enzymolysis temperature is 55 ℃, the fermentation temperature is 32 ℃, and the fermentation time is 24h.
Example 3: preparation of cistanche enzyme for protecting liver and kidney
In this embodiment, on the basis of embodiment 1, a preparation method of cistanche enzyme for protecting liver and kidney is provided, wherein a volume ratio of cistanche to boiling water is 1:7, decocting for 30min; pectinase, cellulase and hemicellulase, wherein the addition amounts of the enzymes are respectively 0.4%, 0.2% and 0.2% of the weight of the cistanche slice, the enzymolysis time is 2.0h, the enzymolysis temperature is 50 ℃, the fermentation temperature is 30 ℃, and the fermentation time is 12h.
Example 4: preparation of cistanche enzyme for protecting liver and kidney
In this embodiment, on the basis of embodiment 1, a preparation method of cistanche enzyme for protecting liver and kidney is provided, wherein a volume ratio of cistanche to boiling water is 1:10, decocting for 60min; pectinase, cellulase and hemicellulase, wherein the addition amounts of the enzymes are respectively 0.8%, 0.4% and 0.4% of the weight of the cistanche slice, the enzymolysis time is 3.5h, the enzymolysis temperature is 60 ℃, the fermentation temperature is 37 ℃, and the fermentation time is 36h.
Example 5: preparation of cistanche enzyme for protecting liver and kidney
In this embodiment, on the basis of embodiment 1, a preparation method of cistanche enzyme for protecting liver and kidney is provided, wherein a volume ratio of cistanche to boiling water is 1:9, decocting for 50min; pectinase, cellulase and hemicellulase, wherein the addition amounts of the enzymes are respectively 0.5%, 0.3% and 0.3% of the weight of the cistanche slice, the enzymolysis time is 3.0h, the enzymolysis temperature is 55 ℃, the fermentation temperature is 35 ℃, and the fermentation time is 26h.
Example 6: optimization of preparation of cistanche enzyme for protecting liver and kidney
In this embodiment, on the basis of embodiments 1 to 5, the process parameters provided by the present invention are subjected to corresponding surface analysis, the effects of the decocting time (a), the enzymolysis time (B), and the fermentation time (C) on the SOD activity (R1) of liver and kidney protecting cistanche enzymes and the sensory score (R2) are mainly considered, a mathematical model of various factors of the preparation process parameters and the quality index of liver and kidney protecting cistanche enzymes is established, and the response surface test factors and the horizontal component attached table 1, and the box-bohnkeb test design and the results are shown in the attached table 2 and the attached tables 1 to 6.
Table 1: response surface test factor level table
| Composition of | Unit of | Minimum value of | Maximum value |
| Decoction time (A) | min | 30 | 50 |
| Time of enzymolysis (B) | h | 2.5 | 3.5 |
| Fermentation time (C) | |
12 | 36 |
Table 2: design result of Box-bohnken experiment
According to response surface data analysis, the optimal process conditions of the cistanche enzyme for protecting liver and kidney provided by the invention are as follows: the decocting time is 40min, the enzymolysis time is 3.0h, the fermentation time is 24h, and the cistanche enzyme SOD for protecting liver and kidney obtained under the process condition has the highest activity and the highest sensory score. In a verification test, the selection process conditions comprise that the decoction time is 40min, the enzymolysis time is 3.0h, and the fermentation time is 24h, the SOD activity of the obtained cistanche enzyme for protecting liver and kidney is 625.03U/mL, the sensory score is 94.68, the measured value is basically consistent with the predicted value, and the process model has a better effect.
Example 7: application of cistanche enzyme for protecting liver and kidney
In this example, based on examples 1 to 6, the performance of the cistanche ferments for protecting liver and kidney provided by the present invention was tested and compared, and the in vitro liver protecting activity and sensory evaluation of the cistanche ferments provided by the present invention were mainly examined.
1. In-vitro liver protection activity of cistanche enzyme
(1) Cistanche enzyme sample cytotoxicity test on WRL68 hepatocytes
Logarithmic phase WRL68 hepatocytes were seeded in 96-well plates (1X 10) 5 cells/mL, 100. Mu.L per well), after culturing for 24h, adding 100. Mu.L LPBS buffer per well to the blank group, adding 100. Mu.L culture medium per well to the control group, adding culture medium containing cistanche enzyme with different concentrations (10-250 mL/L) to the experimental group, and continuing culturing for 12h and 24h. The cell viability was determined by MTT assay for 6 replicate wells per group, and the results of this assay are shown in FIG. 7.
As can be seen from the data in FIG. 7, the enzyme samples at the same concentration have no obvious toxic effect on the cells, but have certain proliferation effect on the cells, the proliferation effect on the cells is not great under the concentrations of 10mL/L and 20mL/Lmg/g, the survival rate of the cells is gradually increased under the concentration of 40mL/L-100mL/L, and the proliferation effect of the enzyme samples on the cells can reach 1.6 times under certain concentration.
(2) Repairing effect of cistanche ferment on alcohol-damaged liver cells
The experiment is divided into a blank group, a control group, a model group and an experimental group, and each group is provided with 6 multiple holes. Only 100. Mu.L of the medium was added to each well of the blank group, and 100. Mu.L of the medium was added to the control group and the model group at a density of 1X 10 5 cells/well cell suspension. Placing in an incubator to be cultured for 24h. After the cells adhere to the wall, the culture medium is sucked off, the cells are washed by PBS buffer solution once, 100 mu L of the culture medium is added into each hole of the control group, 100 mu L of the culture medium containing 400mmol/L ethanol is added into each hole of the injury group and the experiment group, after 24 hours of culture, the cells of the control group and the injury group are replaced by fresh culture media, and the culture media containing cistanche ferment with different concentrations are added into the experiment group. After further 24 hours of incubation, cell viability was determined by the MTT method, and the details of the data are shown in FIG. 8.
As can be seen from the data in FIG. 8, the compositional activity rate of the model is 58.82%. When the concentration of the sample is 50-150mL/L, compared with the model group, the cistanche ferment can obviously improve the survival rate of liver cells (P < 0.01). Cell viability did not show concentration dependence. And when the concentration of the cistanche enzyme reaches 150mL/L, the survival rate of the WRL68 cells is reduced from 120.35% to 108.86%. This is because the sample favors hepatocyte survival at certain concentrations, but cell viability is inhibited when the concentration is high enough to affect the cellular microenvironment. In conclusion, when the concentration of the cistanche ferment sample is 50-150mL/L, the difference is obvious compared with the model group, and the cistanche ferment sample has a certain repairing effect on hepatic cells with alcohol loss, which indicates that the cistanche ferment has an in-vitro liver protection effect.
(3) Mechanism research of repairing effect of cistanche ferment on damaged cells
Cells were plated at 2X 10 6 The cells were seeded in 6-well plates at a density of one cell/mL for 24 hours and cultured for 24 hours, and after cell attachment, the cells were treated according to the method of test 2. After incubation, cells were harvested with 0.05% trypsin-EDTA and disrupted in cold PBS using an ultrasonic cell disruptor to homogenate. The supernatant of the cell lysate was collected and the protein content, MDA, SOD and GSH-Px intracellular activities were determined using the corresponding kit, the specific data are shown in attached Table 3.
As can be seen from the data in Table 3, compared with the negative control group, the activities of SOD and GSH-Px in the model control group are significantly reduced, while the content of MDA is significantly increased; compared with a model control group, after the cells are treated by a 50-150mL/L enzyme sample, the activity of SOD and GSH-PX can be improved, and the content of MDA can be reduced. When the concentration of the sample reaches 100mL/L, the activities of SOD and GSH-PX are respectively increased from (8.45 +/-1.11) U/mgprot and (21.82 +/-1.34) U/mgprot of the model group to (16.17 +/-0.42) U/mgprot and (22.62 +/-0.79) U/mgprot, and the content of MDA is reduced from (1.19 +/-0.15) nmol/mgprot of the model group to (0.72 +/-0.05) nmol/mgprot. The results show that the cistanche ferment repairs the damage of the ethanol-induced cells by increasing the activities of SOD and GSH-PX and reducing the content of MDA.
Table 3: influence of cistanche ferment on activity of SOD, MDA and GSH-Px after WRL68 cell injury induced by ethanol
2. Sensory evaluation test of cistanche enzyme
The cistanche ferments and the non-fermented cistanche stock solution in example 1 were selected for sensory evaluation, and the method was slightly modified with reference to the literature. The sensory evaluation group consisted of 10 trained members, and sensory evaluation was performed on cistanche salsa stock solution and cistanche salsa enzymes in four aspects of smell, taste, color and texture, with a total score of 100 and scoring criteria shown in table 4.
Table 4: sensory evaluation table
The sensory evaluation results are shown in table 5, the total score of the cistanche ferment is 94.00 +/-0.77, the total score of the cistanche stock solution is 84.10 +/-1.14, and the color and luster of the cistanche ferment and the color and luster of the cistanche stock solution are uniform, natural and bright, free of impurity precipitation and uniform in state. After mixed fermentation, the cistanche ferment has pure fermentation flavor, strong aromatic smell, moderate sweet and sour taste, soft and harmonious taste and is obviously superior to cistanche stock solution.
Table 5: sensory evaluation results
| Smell of rice | Acid sweetness | Color | Tissue state | Total score | |
| Cistanche original liquid | 15.78±1.55 | 30.11±1.05 | 14.33±0.70 | 23.78±0.74 | 84.10±1.14 |
| Cistanche enzyme | 18.80±1.03 | 36.70±1.06 | 14.50±0.71 | 24.00±0.82 | 94.00±0.77 |
The data analysis shows that the method takes the cistanche as the raw material, does not contain any additive, adopts a microbial fermentation method to produce the SOD, and has the advantages of easy control of fermentation conditions and short fermentation period. The SOD activity is improved from the original 401.26U/mL to 625.03U/mL, the fermentation improves the flavor of the cistanche ferment, a large amount of organic acid is generated in the fermentation process, so that the cistanche ferment has special sour taste, the whole cistanche ferment is sour, sweet and delicious, the sensory score is improved from 84.10 +/-1.14 to 94.00 +/-0.77, and the cistanche ferment has extremely high edible and health-care values. Therefore, the cistanche ferment has great market potential, and the application range of cistanche is expanded to a certain extent, so that the cistanche which is a valuable resource has more considerable development prospect.
Although lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei adopted in the embodiment are purchased from public sources, if the common culture medium of the six strains is adopted for implementation of the method, fermentation and environmental conditions of cistanche ferment need to be considered, the fermentation control links are many and complex, and the actual situation is difficult to control, the prepared cistanche ferment cannot well reflect the SOD content of the cistanche ferment prepared by the method, and the clearance rate of the prepared cistanche ferment on hydroxyl radicals, the clearance rate of superoxide anions and the clearance rate of DPPH radicals are not as good as those of the unified preparation method provided by the invention.
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.
Claims (8)
1. The cistanche enzyme with the liver and kidney protecting effect is characterized in that the preparation method of the cistanche enzyme specifically comprises the following steps:
(1) Respectively pouring the strain freeze-dried powder of lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei into a conical flask filled with a sterile MRS liquid culture medium on a sterile operation table, sealing the conical flask, placing the conical flask into a constant-temperature incubator at 37 ℃ for activation for 24 to 48 hours to obtain a strain suspension, counting the strain suspension under a microscope by using a blood counting plate, wherein the strain density of the strain suspension is about 10 8 CFU/mL, then continuously culturing 2-3 for replacing for fermentation, and storing the prepared corresponding bacterial suspension in a refrigerated cabinet at 4 ℃ for later use;
(2) Weighing and cleaning the selected cistanche slices, and mixing the materials in percentage by volume: water =1: adding water in a proportion of 7-10, decocting for 30-60 min, cooling and pulping to obtain cistanche salsa slurry;
(3) Taking the cistanche salsa slurry obtained in the step (2), adding 0.4-0.8% of pectinase, 0.2-0.4% of cellulase and 0.2-0.4% of hemicellulase in the weight of cistanche salsa slices, fully stirring, carrying out enzymolysis for 2-3.5 h at 50-60 ℃, and determining the content of soluble solids in cistanche salsa enzymolysis liquid after the enzymolysis reaction is finished; adjusting the sugar degree of the cistanche enzymolysis liquid to ensure that the sugar Brix is 9-13 degrees Bx;
(4) Inactivating enzyme and sterilizing the cistanche enzymolysis liquid obtained in the step (3) at the temperature of 80-100 ℃ for 10-15 min, and then cooling to room temperature for later use;
(5) Inoculating the bacterial suspensions obtained in the step (1) with the inoculum size of 2 x 10 according to a certain volume ratio 6 ～5*10 6 CFU/mL, inoculating the obtained product into the cistanche enzymolysis liquid sterilized in the step (4) for fermentation at the fermentation temperature of 30-37 ℃ for 12-36 h, and measuring the SOD activity after the fermentation is finished, wherein the SOD activity is not lower than 486U/mL;
(6) Filtering the cistanche fermentation liquor obtained in the step (5) at the low temperature of 18-25 ℃, sterilizing at the temperature of 100-121 ℃ for 20-30 min, filling, sealing, packaging and storing at the low temperature to obtain the finished cistanche ferment.
2. The cistanche enzyme for protecting liver and kidney according to claim 1, wherein the bacterial suspension is obtained by mixing 30% -34%, 14% -18%, 15% -19%, 10% -14%, 12% -16% and 7% -11% by volume of lactobacillus reuteri, pediococcus pentosaceus, streptococcus thermophilus, bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei, and the viable count of the bacterial suspension is greater than or equal to 10 8 CFU/mL。
3. The liver and kidney protecting cistanche ferment of claim 1, wherein the culture time of the strain is 36h, and the culture temperature is 37 ℃.
4. The liver and kidney protecting cistanche ferment of claim 1, wherein the volume ratio of cistanche to decocting water is 1:8, decocting for 40min.
5. The cistanche enzyme for protecting liver and kidney according to claim 1, wherein the enzymes are pectinase, cellulase and hemicellulase, the addition amounts of the enzymes are 0.6%, 0.3% and 0.3% of the weight of cistanche slices respectively, the enzymolysis time is 3.0h, and the enzymolysis temperature is 55 ℃.
6. The liver and kidney protecting cistanche ferment of claim 1, wherein the fermentation temperature is 32 ℃ and the fermentation time is 24 hours.
7. The cistanche deserticola ferment for protecting liver and kidney according to claim 1, wherein the sterilization and enzyme deactivation temperature is 90 ℃ and the sterilization and enzyme deactivation time is 12min.
8. The use of the cistanche ferment as claimed in claim 1 for liver and kidney protection.
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| CN116870053A (en) * | 2023-06-20 | 2023-10-13 | 山东第一医科大学附属省立医院(山东省立医院) | Medicine for improving sexual function and preparation method thereof |
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